non-heritable gene editing
CRISPR-Based Anti-Viral Therapy Could One Day Foil the Flu—and COVID-19
Posted on by Dr. Francis Collins
CRISPR gene-editing technology has tremendous potential for making non-heritable DNA changes that can treat or even cure a wide range of devastating disorders, from HIV to muscular dystrophy Now, a recent animal study shows that another CRISPR system—targeting viral RNA instead of human DNA—could work as an inhaled anti-viral therapeutic that can be preprogrammed to seek out and foil potentially almost any flu strain and many other respiratory viruses, including SARS-CoV-2, the coronavirus that causes COVID-19.
How can that be? Other CRISPR gene-editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome to cut out, replace, or repair disease-causing mutations. This new anti-viral CRISPR system also relies on guide RNA. But the guide instead directs a different bacterial enzyme, called Cas13a, to the right spot in the viral genome to bind and cleave viral RNA and stop viruses from replicating in lung cells.
The findings, recently published in the journal Nature Biotechnology [1], come from the lab of Philip Santangelo, Georgia Institute of Technology and Emory University, Atlanta. Earlier studies by other groups had shown the potential of Cas13 for degrading the RNA of influenza viruses in a lab dish [2,3]. In this latest work, Santangelo and colleagues turned to mice and hamsters to see whether this enzyme could actually work in the lung tissue of a living animal.
What’s interesting is how Santangelo’s team did it. Rather than delivering the Cas13a protein itself to the lungs, the CRISPR system works by supplying a messenger RNA (mRNA) with the instructions to make the anti-viral Cas13a protein. This is the same idea as the Pfizer and Moderna mRNA-based COVID-19 vaccines, which temporarily direct your muscle cells to produce viral spike proteins that launch an immune response. In this case, the lung cells translate the Cas13a mRNA to produce the protein. Directed by the guide RNA that was also delivered to the same cells, Cas13a degrades the viral RNA and stops the infection. Because mRNA doesn’t enter the cell’s nucleus, it doesn’t interact with DNA and raise potential concerns about causing unwanted genetic changes.
The researchers designed guide RNAs that were specific to a shared, highly conserved portion of influenza viruses involved in replicating their genome and infecting other cells. They also designed another set directed to key portions of SARS-CoV-2.
Next, they delivered the Cas13a mRNA and guides straight to the lungs of animals using an adapted nebulizer, just like those used to deliver medicines to the lungs of people. In mice with influenza, Cas13a degraded influenza RNA in the lungs and the animals recovered without any apparent side effects. In SARS-CoV-2-infected hamsters, the same approach limited the virus’s ability to replicate in cells as the animals COVID-19-like symptoms improved.
The findings are the first to show that mRNA can be used to express the Cas13a protein in living lung tissue, not just in cells in a dish. It’s also the first to show that the bacterial Cas13a protein is effective at slowing or stopping replication of SARS-CoV-2. The latter raises hope that this CRISPR system could be quickly adapted to fight any future novel coronaviruses that develop the ability to infect humans.
The researchers report that this approach has potential to work against the vast majority—99 percent—of the flu strains that have circulated around the world over the last century. It also should be equally effective against the new and more contagious variants of SARS-CoV-2 now circulating around the globe. While more study is needed to understand the safety of such an anti-viral approach before trying it in humans, what’s clear is basic research advances like this one hold great potential for helping us to fight life-threatening respiratory viruses of the past, present, and future.
References:
[1] Treatment of influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents. Blanchard EL, Vanover D, Bawage SS, Tiwari PM, Rotolo L, Beyersdorf J, Peck HE, Bruno NC, Hincapie R, Michel F, Murray J, Sadhwani H, Vanderheyden B, Finn MG, Brinton MA, Lafontaine ER, Hogan RJ, Zurla C, Santangelo PJ. Nat Biotechnol. 2021 Feb 3. [Published online ahead of print.]
[2] Programmable inhibition and detection of RNA viruses using Cas13. Freije CA, Myhrvold C, Boehm CK, Lin AE, Welch NL, Carter A, Metsky HC, Luo CY, Abudayyeh OO, Gootenberg JS, Yozwiak NL, Zhang F, Sabeti PC. Mol Cell. 2019 Dec 5;76(5):826-837.e11.
[3] Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Abbott TR, Dhamdhere G, Liu Y, Lin X, Goudy L, Zeng L, Chemparathy A, Chmura S, Heaton NS, Debs R, Pande T, Endy D, La Russa MF, Lewis DB, Qi LS. Cell. 2020 May 14;181(4):865-876.e12.
Links:
COVID-19 Research (NIH)
Influenza (National Institute of Allergy and Infectious Diseases/NIH)
Santangelo Lab (Georgia Institute of Technology, Atlanta)
Experts Conclude Heritable Human Genome Editing Not Ready for Clinical Applications
Posted on by Dr. Francis Collins
We stand at a critical juncture in the history of science. CRISPR and other innovative genome editing systems have given researchers the ability to make very precise changes in the sequence, or spelling, of the human DNA instruction book. If these tools are used to make non-heritable edits in only relevant tissues, they hold enormous potential to treat or even cure a wide range of devastating disorders, such as sickle cell disease, inherited neurologic conditions, and muscular dystrophy. But profound safety, ethical, and philosophical concerns surround the use of such technologies to make heritable changes in the human genome—changes that can be passed on to offspring and have consequences for future generations of humankind.
Such concerns are not hypothetical. Two years ago, a researcher in China took it upon himself to cross this ethical red line and conduct heritable genome editing experiments in human embryos with the aim of protecting the resulting babies against HIV infection. The medical justification was indefensible, the safety issues were inadequately considered, and the consent process was woefully inadequate. In response to this epic scientific calamity, NIH supported a call by prominent scientists for an international moratorium on human heritable, or germline, genome editing for clinical purposes.
Following on the heels of this unprecedented ethical breach, the U.S. National Academy of Sciences, U.S. National Academy of Medicine, and the U.K. Royal Society convened an international commission, sponsored by NIH, to conduct a comprehensive review of the clinical use of human germline genome editing. The 18-member panel, which represented 10 nations and four continents, included experts in genome editing technology; human genetics and genomics; psychology; reproductive, pediatric, and adult medicine; regulatory science; bioethics; and international law. Earlier this month, this commission issued its consensus study report, entitled Heritable Human Genome Editing [1].
The commission was designed to bring together thought leaders around the globe to engage in serious discussions about this highly controversial use of genome-editing technology. Among the concerns expressed by many of us was that if heritable genome editing were allowed to proceed without careful deliberation, the enormous potential of non-heritable genome editing for prevention and treatment of disease could become overshadowed by justifiable public outrage, fear, and disgust.
I’m gratified to say that in its new report, the expert panel closely examined the scientific and ethical issues, and concluded that heritable human genome editing is too technologically unreliable and unsafe to risk testing it for any clinical application in humans at the present time. The report cited the potential for unintended off-target DNA edits, which could have harmful health effects, such as cancer, later in life. Also noted was the risk of producing so-called mosaic embryos, in which the edits occur in only a subset of an embryo’s cells. This would make it very difficult for researchers to predict the clinical effects of heritable genome editing in human beings.
Among the many questions that the panel was asked to consider was: should society ever decide that heritable gene editing might be acceptable, what would be a viable framework for scientists, clinicians, and regulatory authorities to assess the potential clinical applications?
In response to that question, the experts replied: heritable gene editing, if ever permitted, should be limited initially to serious diseases that result from the mutation of one or both copies of a single gene. The first uses of these technologies should proceed incrementally and with extreme caution. Their potential medical benefits and harms should also be carefully evaluated before proceeding.
The commission went on to stress that before such an option could be on the table, all other viable reproductive possibilities to produce an embryo without a disease-causing alteration must be exhausted. That would essentially limit heritable gene editing to the exceedingly rare instance in which both parents have two copies of a recessive, disease-causing gene variant. Or another quite rare instance in which one parent has two copies of an altered gene for a dominant genetic disorder, such as Huntington’s disease.
Recognizing how unusual both scenarios would be, the commission held out the possibility that some would-be parents with less serious conditions might qualify if 25 percent or less of their embryos are free of the disease-causing gene variant. A possible example is familial hypercholesterolemia (FH), in which people carrying a mutation in the LDL receptor gene have unusually high levels of cholesterol in their blood. If both members of a couple are affected, only 25 percent of their biological children would be unaffected. FH can lead to early heart disease and death, but drug treatment is available and improving all the time, which makes this a less compelling example. Also, the commission again indicated that such individuals would need to have already traveled down all other possible reproductive avenues before considering heritable gene editing.
A thorny ethical question that was only briefly addressed in the commission’s report is the overall value to be attached to a couple’s desire to have a biological child. That desire is certainly understandable, although other options, such an adoption or in vitro fertilization with donor sperm, are available. This seems like a classic example of the tension between individual desires and societal concerns. Is the drive for a biological child in very high-risk situations such a compelling circumstance that it justifies asking society to start down a path towards modifying human germline DNA?
The commission recommended establishing an international scientific advisory board to monitor the rapidly evolving state of genome editing technologies. The board would serve as an access point for scientists, legislators, and the public to access credible information to weigh the latest progress against the concerns associated with clinical use of heritable human genome editing.
The National Academies/Royal Society report has been sent along to the World Health Organization (WHO), where it will serve as a resource for its expert advisory committee on human genome editing. The WHO committee is currently developing recommendations for appropriate governance mechanisms for both heritable and non-heritable human genome editing research and their clinical uses. That panel could issue its guidance later this year, which is sure to continue this very important conversation.
Reference:
[1] Heritable Human Genome Editing, Report Summary, National Academy of Sciences, September 2020.
Links:
“Heritable Genome Editing Not Yet Ready to Be Tried Safely and Effectively in Humans,” National Academies of Sciences, Engineering, and Medicine news release, Sep. 3, 2020.
International Commission on the Clinical Use of Human Germline Genome Editing (National Academies of Sciences, Engineering, and Medicine/Washington, D.C.)
Video: Report Release Webinar , International Commission on the Clinical Use of Human Germline Genome Editing (National Academies of Sciences, Engineering, and Medicine)
National Academy of Sciences (Washington, D.C.)
National Academy of Medicine (Washington, D.C.)
The Royal Society (London)
Nano-Sized Solution for Efficient and Versatile CRISPR Gene Editing
Posted on by Dr. Francis Collins
Credit: Guojun Chen and Amr Abdeen, University of Wisconsin-Madison
If used to make non-heritable genetic changes, CRISPR gene-editing technology holds tremendous promise for treating or curing a wide range of devastating disorders, including sickle cell disease, vision loss, and muscular dystrophy. Early efforts to deliver CRISPR-based therapies to affected tissues in a patient’s body typically have involved packing the gene-editing tools into viral vectors, which may cause unwanted immune reactions and other adverse effects.
Now, NIH-supported researchers have developed an alternative CRISPR delivery system: nanocapsules. Not only do these tiny, synthetic capsules appear to pose a lower risk of side effects, they can be precisely customized to deliver their gene-editing payloads to many different types of cells or tissues in the body, which can be extremely tough to do with a virus. Another advantage of these gene-editing nanocapsules is that they can be freeze-dried into a powder that’s easier than viral systems to transport, store, and administer at different doses.
In findings published in Nature Nanotechnology [1], researchers, led by Shaoqin Gong and Krishanu Saha, University of Wisconsin-Madison, developed the nanocapsules with specific design criteria in mind. They would need to be extremely small, about the size of a small virus, for easy entry into cells. Their surface would need to be adaptable for targeting different cell types. They also had to be highly stable in the bloodstream and yet easily degraded to release their contents once inside a cell.
After much hard work in the lab, they created their prototype. It features a thin polymer shell that’s easily decorated with peptides or other ingredients to target the nanocapsule to a predetermined cell type.
At just 25 nanometers in diameter, each nanocapsule still has room to carry cargo. That cargo includes a single CRISPR/Cas9 scissor-like enzyme for snipping DNA and a guide RNA that directs it to the right spot in the genome for editing.
In the bloodstream, the nanocapsules remain fully intact. But, once inside a cell, their polymer shells quickly disintegrate and release the gene-editing payload. How is this possible? The crosslinking molecules that hold the polymer together immediately degrade in the presence of another molecule, called glutathione, which is found at high levels inside cells.
The studies showed that human cells grown in the lab readily engulf and take the gene-editing nanocapsules into bubble-like endosomes. Their gene-editing contents are then released into the cytoplasm where they can begin making their way to a cell’s nucleus within a few hours.
Further study in lab dishes showed that nanocapsule delivery of CRISPR led to precise gene editing of up to about 80 percent of human cells with little sign of toxicity. The gene-editing nanocapsules also retained their potency even after they were freeze-dried and reconstituted.
But would the nanocapsules work in a living system? To find out, the researchers turned to mice, targeting their nanocapsules to skeletal muscle and tissue in the retina at the back of eye. Their studies showed that nanocapsules injected into muscle or the tight subretinal space led to efficient gene editing. In the eye, the nanocapsules worked especially well in editing retinal cells when they were decorated with a chemical ingredient known to bind an important retinal protein.
Based on their initial results, the researchers anticipate that their delivery system could reach most cells and tissues for virtually any gene-editing application. In fact, they are now exploring the potential of their nanocapsules for editing genes within brain tissue.
I’m also pleased to note that Gong and Saha’s team is part of a nationwide consortium on genome editing supported by NIH’s recently launched Somatic Cell Genome Editing program. This program is dedicated to translating breakthroughs in gene editing into treatments for as many genetic diseases as possible. So, we can all look forward to many more advances like this one.
Reference:
[1] A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing. Chen G, Abdeen AA, Wang Y, Shahi PK, Robertson S, Xie R, Suzuki M, Pattnaik BR, Saha K, Gong S. Nat Nanotechnol. 2019 Sep 9.
Links:
Somatic Cell Genome Editing (NIH)
Saha Lab (University of Wisconsin-Madison)
Shaoqin (Sarah) Gong (University of Wisconsin-Madison)
NIH Support: National Eye Institute; National Institute of General Medical Sciences; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; Common Fund
